Association of the Dopamine Transporter Gene (DAT1) With Poor Methylphenidate Response

Association of the Dopamine Transporter Gene (DATl) With Poor Methylphenidate Response BERTRAND G. WINSBERG, M.D., AND DAVID E. COMINGS, M.D.

ABSTRACT Objective: This study attempted to relate the alleles of the D2( D R M ) ,D4(DRD4),and dopamine transporter (DAT7)genes to the behavioral outcome of methylphenidate therapy. Method: African-Americanchildren with attention-deficithyperactivity disorder were treated with methylphenidate in doses not in excess of 60 mg/day.The dosage was increased until behavioral change was achieved, using a decrement in scores of less than or equal to 1 on a commonly used rating scale or until the maximum tolerated dose was achieved. Blood samples were obtained at that point, and genotypes for polymorphism at the respective genes were identified. Results: Genotypes were then tested by

x2 to assess the significance of any associ-

ation with drug response. Only the dopamine transporter gene was found to be significant. Homozygosity of the 10-repeat allele was found to characterize nonresponse to methylphenidate therapy ( p = ,008). Conclusions: While the results suggest that alleles of the dopamine transporter gene play a role in methylphenidate response, replication in additional studies is needed. J. Am. Acad. Child Adolesc. Psychiatry, 1999, 38(12):1474-1477. Key Words: attention-deficithyperactivity disorder, drug response, dopamine transporter, molecular genetics.

Attention-deficit hyperactivity disorder (ADHD) is a complex neurobehavioral disorder. Twin studies indicate that the heritability is 70% to 95% (Gillis et al., 1992; Sherman et al., 1997a,b; Stevenson, 1992; Thapar et al., 1995). Like most behavioral disorders, it is probably polygenic (Comings, 1996; Comings et al., 1996, 1998), i.e., due to the additive and interactive effect of multiple genes, each with a small effect. Association studies have implicated a number of dopaminergic and noradrenergic genes in ADHD alone and A D H D with chronic tics. These include the dopamine receptor genes DRD2 (Comings, 1996; Comings et al., 1991), DRD4 (Comings et al., 1999; LaHoste et al., 1996; Rowe et al., 1998), and DRD5 (Comings et al., 1998; Daly et al., 1999), DATl (Comings et al., 1996; Cook et al., 1995; Daly et al., 1999; Gill et al., 1997; Waldman et al., 1998), dopamine pAccepted June 22, 1999. DT: Winsberg is with the Division of Child Psychiaq and Pediatrics, Brookdale Universiq Hospitul and Medical Center, Brooklyn, M and Research Associate, Nathan Kline Institute, Orangeburg,NY Dr. Comings is with the Department of Medical Genetics, Ciq of Hope National Medical Center, Duarte, CA. Supported by the Mary Ellen Gerber Foundation. Reprint requests to Dr. Winsberg,Division of ChildPsychiany and Pediatrics, Brookdale Uniuersiy Hospital and Medical Center, I Brookdale Plaza, 12 CHC Brooklyn, N Y II212. 0890-8567/99/3812-14740 1999 by the American Academy of Child and Adolescent Psychiatry

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hydtoxylase (DBH) (Comings et al., 1996, 1999; Daly et al., 1999),2 adrenetgic genes, ADRA2A and ADRA2C (Comings et al., 1999), the androgen receptor gene (AR), and other genes (Comings et al., 1998). DATl is a particularly relevant candidate gene for ADHD because the dopamine transporter is the site of action of the stimulant medications (methylphenidate, dextroamphetamine, pemoline, bupropion) used in the treatment of ADHD (Seeman and Madras, 1998). In addition, knockout mice, missing this gene, are extremely hyperactive (Caron, 1996; Giros et al., 1996). Because the evolving evidence suggests that polymorphisms of the DRD2 and DRD4 genes may also be implicated in ADHD, it seemed reasonable to also investigate their polymorphisms with respect to stimulant response. In addition to understanding their etiology, one of the additional benefits of studies of the molecular genetics of behavioral disorders is the potential to identify those individuals who are most likely to respond to specific types of medication. To our knowledge, the first study of this type in relation to dopaminergic genes was by Lawford et al. (1995). Their data suggested that alcoholics carrying the 22 genotype of the Zq I A polymorphism of the DRD2 gene responded to treatment with bromocriptine, whereas those carrying the 1 allele did not. In a recent study, using the same polymorphism, we found that

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obese subjects carrying the 22 genotype responded to treatment with chromium picolinate whereas those carrying the 1 allele did not (unpublished). Similar treatment response studies have been reported for other genes including the H T E A gene for clozapine response in schizophrenia (Arranz et al., 1995) and the serotonin transporter gene (HTT) for response to fluvoxamine (Smeraldi et al., 1998). In a report on the DRD4 receptor polymorphism, LaHoste et al. (1996) reported that 49% of the ADHD children in their sample had the 7-repeat allele as contrasted to 2 I Yo of controls. This report has received support from other studies (Rowe et al., 1998) but not all (Lau et al., 1997). Winsberg and colleagues have previously conducted studies in an effort to explain the response to stimulant treatment among hyperkinetic children. In addition to electrophysiological methods (Winsberg et al., 1997), they also used pharmacokinetic approaches (Hungund et al., 1978; Richardson et al., 1988; Winsberg et al., 1982, 1987). However, results have been inconclusive. O n the basis of the advances reviewed above, we felt that a molecular genetics approach would be a promising approach to exploring the possible differences between methylphenidate (MPH) responders and nonresponders. We report our preliminary observations on the potential association between polymorphisms of the DRD2, DRD4, and DATI genes and MPH response in African-American children with ADHD. METHOD

teachers. We elected to use increments in the oral dose to elicit the response in keeping with standard clinical protocol management procedures and with our previous work, which has shown oral dose to be as adequate a predictor of clinical response as milligram-per-kilogram dose assignment and plasma MPH concentrations (Richardson et al., 1988). The dose was restricted to no more than 60 mglday but not to exceed 0.7 mg/kg in keeping with side effects in doses that approach this ceiling (Winsberg et al., 1982). No nonresponder received less than 40 mg/day. Blood samples were obtained at the time of the establishment of the response criterion and after the parent gave written authorization. Specimens were sent to the Department of Medical Genetics at the City of Hope National Medical Center for genotyping. The study was approved by the institutional review boards of both institutions, and parents provided written informed consent. It was our intent to gather data on 30 children to determine whether this approach to the study of nonresponse would prove encouraging to further investigation.

Genotyping

All genotyping was performed by polymerase chain reaction-based methods. For the DRD2 gene we used the Tag I Al/A2 polymorphism 3' to the gene described by Grandy et al. (1989). For the DRD4 gene we used the 48 bp repeat polymorphism in the coding region of the third intracytoplasmic loop described by VanTol et al. (1992). For the DATI gene we used the polymorphism described by Vandenbergh et al. (1992). Statistics The response of the ABRS to MPH treatment was evaluated using paired t tests for baseline versus treatment scores. This analysis was conducted to confirm that we had achieved our response criterion. Once we had our data, they were reviewed and the distribution of values seemed appropriate to the application of analysis. We then tested for the associations between the genotypes in the responders versus nonresponders. All analyses were conducted with the Primer statistical program (McGraw-Hill Inc., New York).

xz

RESULTS

Subjects Subjects were drawn from the clinic samples under the first author's care at Brookdale Hospital University Medical Center in Brooklyn, New York. Subjects were children with a diagnosis of ADHD based on DSM-IZZ-R criteria as described in previous reports (Winsberg et al., 1982, 1997). All subjects were African-American and had no prior stimulant therapy. Subjects with comorbid conduct disorder and mental retardation (IQ 5 70) were excluded. I Q was assessed by standard but varied clinical tests. Children were considered eligible for pharmacotherapy with MPH if they achieved a score of greater than 1.5 on the widely used Conners Abbreviated Rating Scale (ABRS) (Conners and Barkley, 1985). This instrument yields mean scores ranging from 0 (no deviance) to 3 (maximum deviance). Children were then treated to achieve their best response status using the ABRS as the guide to dose increment and parent and teacher information regarding side effects. Responders and nonresponders were established as in previous studies (Winsberg et al., 1997). A clinical response to medication was defined as a decrement in the mean score from greater than 1.5 to equal to or less than 1.0 on 2 consecutive ABRS ratings during treatment with MPH. Nonresponders were defined as children who did not achieve a decrement to 1 or less. Children received MPH in weekly dose increments; their response was monitored with scale scores obtained from their

Subjects

The sample consisted of 16 responders and 14 nonresponders. Responders ranged in age from 7 to 11 years (mean 9.01 f 1.86 years). Nonresponders ranged in age from 6 to 9.4 years (mean 7.5 1 f 1.38 years). The baseline ABRS for the 16 responders ranged from 1.5 to 2.9 (mean 2.37 f 0.41) and for the 14 nonresponders from 1.7 to 3.0 (mean 2.72 f 0.31). The posttreatment mean ABRS score was 0.73 -+ 0.33 for the responders and 2.61 * 0.39 for the nonresponders, reflecting the response criterion for the study. Paired t test comparison between baseline and treatment ABRS for the responder group indicated a significant decrement in scores (t = 17.33,p < .OO 1). The difference between responders and nonresponders at posttreatment was significant as expected ( t = 14.33, df = 29, p < .OOl), confirming that our treatment criteria had been met.

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WINSBERG AND C O M I N G S Genotypes

With respect to the DRD2 gene there were only 2 genotypes, 12 and 22. Of the total group, 50% (1513O)were heterozygous for the 1 allele of the Taq I A polymorphism. Among the good responders, 56.2% (9/16) were heterozygotes compared with 42.8% (6/14) of the poor responders. These were not significantly different. With respect to the DRD4gene, 30% (9130) of our sample carried the 7 allele of the DRD4 polymorphism compared with 49% in the predominantly (85%) white subjects reported by LaHoste and colleagues (1996). Among the good responders, 37.5% (6/16) carried the 7 allele compared with 21.4% of the poor responders. These differences were not significant. Only the DATI gene showed significant differences (Table 1).There was a significant increase in the frequency of individuals homozygous for the 10 copy allele in the nonresponders (86%) compared with responders (31Yo) ( p = .OO8). The significance persisted with a Bonferroni corrected a = .05/3 or .O17. DISCUSSION

The finding of a significant relationship between 10/ 10 homozygosity and nonresponse is consistent with the evidence implicating the dopamine transporter as the site of action of MPH. Seeman and Madras (1998) reviewed the relationship of the DATI and stimulant response. Stimulants bind to and inhibit the reuptake function of the transporter. As the 1O/1O allele is associated with nonresponders, we can speculate that binding of MDH to the dopamine transporter might be different from that of those not carrying the 10/10 genotype and that homozygosity at this polymorphism is more severe in this regard than heterozygosity. This observation can lead to a testable hypothesis using a cloned DATI gene to assess nonresponders' physiological response to MPH. A cautionary note, however, is warranted. Gainetdinov et al. (1999) recently provided evidence that mice with hyperactivity consequent to a genetically engineered absence of the transporter also show a diminution in hyperactivity in a TABLE 1 DATI Genotype by Treatment Response Responders

Nonresponders

5 (31)

12 (86) 2

10/10 allele, no. (%) 9/10: 8/10: 5/9 allele Note:

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x2 = 6.938, df= 1, p

11 =

.oo8.

dose-dependent manner when treated with MPH. An attenuation of hyperactivity was also found with compounds which enhance serotonergic neurotransmission (e.g., fluoxetine and 5-hydroxytryptophan). The role of serotonin neurotransmission is thus open as an explanatory notion in the etiology and treatment of animal models of hyperactivity. The DATZ gene has been associated with ADHD in a number of clinical studies (Comings et al., 1996; Cook et al., 1995; Daly et al., 1999; Gill et al., 1997; Waldman et al., 1998).Waldman et al. provide evidence implicating DATI in ADHD by corroborating the between-family association of a significant association between high-risk alleles and hyperactivity symptoms and extend this by showing that siblings with the greater number of highrisk alleles had greater deviance scores. They report no DATI ethnically based differences in their sample. We note that our African-American children are in general poor responders to stimulants. Although our subjects were not specificaliy selected to be a representative sample of our population, only 53% of our subjects were responders to MPH. This is in contrast to the expected 70% to 80% response rate for predominantly white samples. While we are acutely aware of the problems pertaining to racial identification in biological research (cf. La Veist, 1996), our data require some discussion relative to our findings and those of other investigators. The clinic population from which the sample was obtained is almost solely African-American and is representative of the population served by the hospital. Inasmuch as Waldman et al. (1998) found no ethnic differences in their sample, it would seem that differentia1 representation of high-risk alleles as a function of ethnicity would not likely account for any possible difference in etiologies of hyperkinesis. Their report, which used linkage-based strategies, was not treatment-focused and consequently could not address the issue of differential drug response. Finally, it has been proposed that a better knowledge of genetic variation may lead to innovative pharmaceuticals (Kleyn and Vessell, 1998). This speculation may be actualized in pediatric psychopharmacology should future research assist in the establishment of the molecular genetic basis of psychostimulant response. Limitations

Despite the increasingly compelling evidence implicating the DATI in ADHD and our data suggesting its role

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in treatment response, caution is required. Replication both in African-Americans and whites is needed because Our findings are based on a small sample size with only one outcome measure. Nevertheless, the present results should now allow for more precise hypothesis generation in hture research. REFERENCES hranz M, Collier D, Sodhi M et al. (1995), Association between clozapine response and allelic variation in 5-HT2A receptor gene. Lancet 346:28l-282 Caron M (1996), A mouse knockout. Am JPsychiatry 153:1387 Comings DE (1996), Polygenetic inheritance of psychiatric disorders. In: Handbook ofPychiatric Genetics, Blum K, Noble EP, Sparks RS, Sheridan eds. Boca Raton, FL: CRC Press, pp 235-260 Comings DE, Comings BG, Muhleman D et al. (1991), The dopamine D2 receptor locus as a modifying gene in neuropsychiatric disorders. ] M A 266:1793-1800 Comings DE, Gade-Andavolu R, Gonzalez N et al. (1998), Multiple additive associations: a technique for the identification of genes in polygenic disorders. Am JMed Genet 81:489 Comings DE, Gade-Andavolu R, Gonzalez N, Blake H, Wu S, MacMurray JP (1999), Additive effect of three adrenergic genes ( A D M A , A D M C , DBH) on ADHD in subjects with Tourette syndrome with and without learning disabilities. Clin Genet 55:160-172 Comings DE, Wu H, Chiu C, Ring RH, Dietz G, Muhleman D (1996), Polygenic inheritance of Tourette syndrome, stuttering, ADHD, conduct and oppositional defiant disorder: the additive and subtractive effect of the three dopaminergic genes-DRD2, DBH and DATl. Am JMed Genet 67:264-288 Conners CK, Barkley RA (ISSS), Rating scales and checklisrs for child psychopharniacology. Pychopharmacol Bull 21 :809-843 Cook EH, Stein MA, Krasowski M D et al. (1995), Association of attentiondeficit disorder and the dopamine transporter gene. Am J Hum Genet 56:993-998 Daly G, Hawi 2, Fitzgerald M, Gill M (1999), Mapping susceptibility loci in attention deficit hyperactivity disorder: preferential transmission of parental alleles at DAT1, DBH and DRD5 to affected children. Mol Psychiatry 4:192-196 Gainetdinov RR, Wetsel WC, Jones SR, Levin ED, Jaber M, Caron MG (1999), Role of serotonin in the paradoxical calming effect of psychostimulants on hyperactivity. Science 283:397-402 Gill M, Daly G, Heron S, Hawi 2,Fitzgerald M (1997), Confirmation of association between attention deficit disorder and a dopamine transporter polymorphism. Mol Psychiatry 2:311-313 Gillis JJ, Gigler JW, Pennington BF, DeFries JC (1992), Attention deficit disorder in reading-disabled twins: evidence for a genetic etiology. JAbnorm Child Psychol 20:343-348 Giros B, Jaber M, Jones SR, Wightman RM, Caron MG (1996), Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking dopamine transporter. Nature 379:606-6 12 Grandy DK, Litt M, Allen L et al. (1989), The human dopamine D2 receptor gene is located on chromosome 11 at q22-q23 and identifies a Taq IRFLl? Am J Hum Genet 45:778-785 Hungund BL, Hanna M, Winsberg BG (1978),A sensitive gas chromatographic method for the determination of methylphenidate (Ritalin) and its major

metabolite alpha-phenyl-2-piperidine acetic acid (ritalinic acid) in human plasma using nitrogen-phosphorous detector. Commun Psychophanacol 2:203-208 Kleyn PW, Vessel1 ES (1998), Generic variation as a guide to drug development. Science 281:1820-1821 La Veist TA (1996), Why we should continue to study race . . . but do a better job: an essay on race, racism and health. Ethn Dis 6:21-29 LaHoste GJ, Swanson JM, Wigal SB et al. (1996), Dopamine D4 receptor gene polymorphism is associated with attention deficit hyperactivity disorder. Mol Pjychiatry 1: 121-124 Lau EK, Castellanos FK, Tayebi N et al. (1997), Association studies do not show an increased frequency of the 7-fold repeat form of the dopamine 4 receptor polymorphisms in children with attention deficit disorder (ADHD) (abstract). Am J H u m Genet 61:A203 Lawford BR, Young DMCD, Rowell JA et al. (1995), Bromocriptine in the treatment of alcoholics with the D2 dopamine receptor A1 allele. NatMed 1:337-341 Richardson E, Kupietz S, Winsberg BG, Maitinsky S, Mendell N (1988), Effects of methylphenidate dosage in hyperactive reading-disabled children, 11: reading achievement. / A m Acad ChildAdohsc Psychiatry 2778-87 Rowe DC, Stever C, Giedinghagen LN et al. (1998), Dopamine DRD4 receptor polymorphism and attention deficit hyperactivity disorder. Mol Psychiatry 3:419-426 Seeman P, Madras BK (1998), Anti-hyperactivity medication: methylphenidate and amphetamine. Mol Psychiatry 3:386-396 Sherman DK, Iacono WG, McGue MK (1997a), Attention-deficit hyperactivity disorder dimensions: a twin study of inattention and impulsivityhyperactivity. / A m Arad ChildAdolesc Psychiatry 36:745-753 Sherman DK, McGure MK, Iacono WG (1997b), Twin concordance for attention deficit hyperactivity disorder: a comparison of teachers’ and mothers’ reports. Am J Pychiaty 154:532-535 Smeraldi E, Zanardi R, Benedetti F, Di Bella D, Perez J, Catalan0 M (1998), Polymorphism within the promoter of the serotonin transporter gene and antidepressant efficacy of fluvoxamine. Mol Psychiatry 3: 508-5 11 Stevenson J (1992), Evidence for a genetic etiology in hyperactivity. Behav Genet 22:337-344 Thapar A, Hervas A, McGuffin I‘ (1995), Childhood hyperactivity scores are highly heritable and show sibling competition effects: twin study evidence. Behav Genet 25:537-544 Vandenbergh DJ, Persico AM, Ulh GR (1992), A human dopamine transporter predicts reduced glycosylation, displays a novel repetitive element and provides racially-dimorphic Taq IRFL Ps. Mol Brain Res 15:161-166 VanTol HHM, Wu CM, Guan H-C et al. (1992), Multiple dopamine D4 receptor variants in human population. Nature 358:149-152 Waldman ID, Rowe A, Abramowitz S et al. (1998), Association and linkage of the dopamine transporter gene and attention-deficit hyperactivity disorder in children: heterogeneity owing to diagnostic subtype and severity. Am J Hum Genet 63: 1767-1776 Winsberg B, Maitinsky S, Kupietz S, Richardson E (1987), Is there dosedependent tolerance associated with chronic methylphenidate therapy in hyperactive children: oral dose and plasma considerations.Pychopharmacol Bull 23:107-110 Winsberg BG, Javitt DC, Silipo G, Doneshka P (1997), Mismatch negativity in hyperactive children: effects of methylphenidate. Biol Psychiatry 42:423-445 Winsberg BG, Kupietz SS, Sverd J, Hungrund BL, Young NL (1982), Methylphenidate oral dose plasma concentrations and behavior response in children. Psychopharmacology 76:329-332

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